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Soybean mosaic virus sequence capable of inhibiting growth of agrobacteria and application of soybean mosaic virus sequence

A technology of soybean mosaic virus and Agrobacterium, applied in the field of genetic engineering, achieves the effect of remarkable inhibition ability, excellent practical performance and great application value

Active Publication Date: 2018-11-06
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there have been no reports on the interaction between potyviruses and Agrobacterium

Method used

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  • Soybean mosaic virus sequence capable of inhibiting growth of agrobacteria and application of soybean mosaic virus sequence
  • Soybean mosaic virus sequence capable of inhibiting growth of agrobacteria and application of soybean mosaic virus sequence
  • Soybean mosaic virus sequence capable of inhibiting growth of agrobacteria and application of soybean mosaic virus sequence

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1: Discovery of Agrobacterium Growth Inhibitory Sequences in Soybean Mosaic Virus

[0025] 1. Construction of soybean mosaic virus cloning vector

[0026] We used trizol to extract total RNA from fresh SMV-infected soybean leaves; total RNA used TAKARA primescript TM ii 1st strand cDNA synthesis kit reverse transcription into cDNA. Using cDNA as a template, we divided SMV into 5 segments to amplify and purify and recover. The recovered above-mentioned PCR fragments (such as 1a, 2a, 3a, 4a, 5a five fragments) are directly transformed into Clontech's yeast strain Y2Hgold with the pCB301 vector linearized with StuI and SmaI, and use SD / -Trp medium Screen for recombinant clones. The plasmids extracted from single yeast colonies were verified by PCR and sequencing, and then the plasmids were amplified. The successfully constructed virus clones were named pCB301-SC3 (#SC3), pCB301-SC7 (#SC7) and pCB301-SC15 (#SC15), respectively. The strategy for obtaining each P...

Embodiment 2

[0042] Example 2: Application of SC15 Agrobacterium Growth Inhibition Sequence in Efficient Screening of Recombinant Clones

[0043] 1. Construction of pCB301-attL-SC15P vector

[0044] We replaced the sequence between LB and RB of the pCB301 vector with attL1 and attL2 sequences, and inserted a small fragment SC15P that can inhibit the growth of Agrobacterium between the attL1 and attL2 sequences, and the constructed vector was named pCB301-attL-SC15P( image 3 A, the complete sequence of pCB301-attL-SC15P is shown in SEQ ID NO.3). The construction process of pCB301-attL-SC15P is as follows: first, use the pGWB5 plasmid as a template, use primers attL1-301REC and attL2-301REC to perform PCR amplification to obtain fragment 1s, use pCB301 plasmid as a template, and use primers 301-frag-F and 301- Frag-R was amplified by PCR to obtain fragment 2s. The purified 1s and 2s fragments were ligated and transformed into Escherichia coli DH5α through the ClonExpress-II OneStep Cloning...

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Abstract

The invention discloses a soybean mosaic virus sequence capable of inhibiting growth of agrobacteria, and proves that the sequence can be used for directly screening recombinant clones in agrobacteria. The agrobacteria are important host bacteria in a plant transformation system, but the constructed recombinant clones need to be screened in Escherichia coli before being transferred to the agrobacteria. The soybean mosaic virus strain SC15 has the characteristic of inhibiting growth of the agrobacteria, but strains SC3 and SC7 do not have the same effect; by virus sequence truncation experiments, the sequence SC15P associated with growth inhibition of the agrobacteria in SC15 is identified; a vector compatible with a gateway cloning system is constructed by the sequence, LR reaction and transformation experiments are conducted, and finally, the assumption of recombinant clone in the agrobacteria by SC15P is achieved.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a soybean mosaic virus sequence capable of inhibiting the growth of Agrobacterium and its application. Background technique [0002] Agrobacterium is a Gram-negative bacterium belonging to the bacteria kingdom Proteobacteria, Rhizobium order Rhizobiumaceae. Agrobacterium can transfer and insert a part of its own DNA sequence into the DNA of plants, which makes it an important plant transgenic tool and is widely used in genetic transformation and transient expression experiments of plants. However, Agrobacterium is seldom used directly as a host for recombinant clone selection. Recombinant clones used in Agrobacterium infection experiments usually need to be screened and identified in other microorganisms such as Escherichia coli in advance, which not only makes the experimental process too cumbersome, but also increases the time-consuming of the experiment. [0003] Recombinant...

Claims

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Application Information

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IPC IPC(8): C12N15/40C12N15/63
CPCC07K14/005C12N15/63C12N2770/34022C12N2770/34032
Inventor 智海剑王丽群殷金龙刘慧晋彤彤李凯郭东全
Owner NANJING AGRICULTURAL UNIVERSITY
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